Heat-induced Bovine Serum Albumin Research Articles

Experimental procedures to investigate fibrillation of proteins

The unwanted phenomenon of protein fibrillation is observed in vivo and during therapeutic protein development in the industry. Protein aggregation is associated with various degenerative disorders and might induce immune-related challenges post-administration of biopharmaceutics. A pipeline for early detection, identification, and removal of pre-formed fibrils is needed to improve the quality, efficacy, and effectiveness of the formulation. Protein fibril formation is accompanied by unfolding, secondary structural changes and the formation of larger aggregates. However, most detection processes come with extensive sample preparation steps and inefficient repeatability, incurring a financial burden on research. The current article summarizes and critically discusses six simple yet powerful methods to detect aggregation phenomena in the line of detecting fibrillar aggregates in heat-induced bovine serum albumin protein. Comparing the native and heat-induced protein samples would provide insights about aggregates. Easy, inexpensive and optimized protocols for detecting the fibrillation of proteins are explained. The procedures mentioned here detected the appearance of β-sheet-rich fibrils in the heat-induced protein sample. The aggregation is characterized by enhanced thioflavin-T fluorescence, alteration in the intrinsic fluorescence, decrease in helicity and subsequent increase in β-sheet and appearance of particles with larger hydrodynamic diameters.•This article summarizes various analytical techniques to easily characterize the fibrillation of proteins.•Various techniques to detect the formation of β-sheet rich structures, changes in the secondary structures and size of aggregates have been discussed.•The stated methodologies are validated on a model protein, albumin.

A desirability approach for Antidiabetic and Antiinflammatory efficacy of Ficus subincisa bark

Background: This study aimed to examine the anti-inflammatory and antidiabetic effects of the green solvent extract from F.subincisa Buch-Ham.ExSm.This paper also explains how bio-based products have been highly safe and alternative to the use of organic solvent. Methods: The serial extraction was carried out with increasing polarity using a magnetic stirrer: d-limonene, Isopropyl alcohol, Ethyl lactate, and Hydroalcohol.The concentrated and dried extracts were subjected to analysis the antidiabetic activity and anti-inflammatory activity were assessed by employing standard in vitro techniques. Result: In vitro anti-inflammatory assays were performed using heat-induced bovine serum albumin (BSA) denaturation and erythrocyte membrane stabilization assays.α-amylase and α-glucosidase were used to evaluate the anti-diabetic activity of F.subincisa bark in vitro. Diclofenac sodium, Acarbose, and aspirin were used as reference drugs. The in vitro method showed significant anti-inflammatory and anti-diabetic properties of different bio-based extracts tested. Conclusions: Findings from this study showed that F.subincisa exhibited substantial anti-inflammatory and antidiabetic actions in vitro models.

LCMS/MS analysis and evaluation of anti-inflammatory and antioxidant activities of the polyphenol fraction of Litsea quinqueflora (Dennst.) Suresh

The main aim of the work was to scientifically prove the anti-inflammatory property of the polyphenol-rich fraction of hydro-alcoholic leaf extract of Litsea quinqueflora (Dennst.) Suresh by protein denaturation and free radical scavenging activity. The polyphenol-rich fraction of hydro-alcoholic leaf extract was obtained via acid-alkali hydrolysis, followed by fractionation with chloroform and ethyl acetate. HPTLC profiling of the finally obtained ethyl acetate fraction and consequent derivatisation with aluminium chloride revealed the presence of flavonoids in a more purified form. LCMS/MS analysis tentatively identified the presence of bioactive polyphenolic compounds such as gallocatechin, sinapic acid, pinocembrin, paeonol and umbelliferone in the separated fraction. The polyphenol-rich fraction of hydro-alcoholic extract of leaves showed anti-denaturing activity in heat-induced bovine serum albumin denaturation with an IC50 value of 23.59 µg/ml and was statistically significant at 0.1% level. The antioxidant property of the polyphenol-rich fraction determined by its free radical scavenging ability against DPPH and ABTS showed IC50 values 122.98 and 135.44 µg/ml respectively and was also statistically significant at 0.1% level. Hence, the traditional use of Litsea quinqueflora as an anti-inflammatory agent can be attributed to the presence of polyphenols.

Heat-induced self-assembling of BSA at the isoelectric point

Materials based on ordered protein aggregates have recently received a lot of attention for their application as drug carriers, due to their biocompatibility and their ability to sequester many biological fluids. Bovine serum albumin (BSA) is a good candidate for this use due to its high availability and tendency to aggregate and gel under acidic conditions. In the present work, we employ spectroscopic techniques to investigate the heat-induced BSA aggregation at the molecular scale, in the 12–84 °C temperature range, at pH = 5 where two different isoforms of the protein are stable. Samples at low and high protein concentration are examined. With the advantage of the combined use of FTIR and CD, we recognize the aggregation-prone species and the different distribution of secondary structures, conformational rearrangements and types of aggregates, of millimolar compared to micromolar BSA solutions. Further, as a new tool, we use the Maximum Entropy Method to fit the kinetic curves to investigate the distribution of kinetic constants of the complex hierarchical aggregation process. Finally, we characterize the activation energy of the initial self-assembling step to observe that the formation of both small and large aggregates is driven by the same interactions.

Evaluation of anti-inflammatory activity of Justicia secunda Vahl leaf extract using in vitro and in vivo inflammation models

BackgroundJusticia secunda Vahl. is a medicinal plant used in ethnomedical practice as therapy to manage inflammation. Therefore, this study was designed to evaluate the anti-inflammatory activity of methanol extract of J. secunda leaves (MEJSL) using in vitro and in vivo inflammation models.MethodsSeventy-percent MEJSL was prepared following standard procedure. In vitro anti-inflammatory assays were performed using heat-induced bovine serum albumin (BSA) denaturation and erythrocyte membrane stabilization assays. Carrageenan and formaldehyde induced inflammation in rat models were used to evaluate the anti-inflammatory activity of MEJSL in vivo. Diclofenac sodium was used as a reference drug. In addition, liver and kidney function assays and hematological analysis were carried out.ResultsData revealed that varying concentrations of MEJSL significantly (P < 0.05) inhibited heat-induced BSA denaturation and stabilized erythrocyte membrane against hypotonicity-induced hemolysis when compared with diclofenac sodium in a concentration-dependent manner. In vivo study showed that 10 mg/kg body weight (b.w.) diclofenac sodium, 100 and 300 mg/kg b.w. MEJSL suppressed carrageenan-induced paw edema at the sixth hour by 71.14%, 83.08%, and 89.05%, respectively. Furthermore, 10 mg/kg b.w. diclofenac sodium, 100 and 300 mg/kg b.w. MEJSL inhibited formaldehyde-induced paw edema by 72.53%, 74.73%, and 76.48%, respectively. Animals treated with varying doses of MEJSL had reduced plasma aspartate aminotransferase and alanine aminotransferase activities; urea and creatinine concentrations; and modulated hematological parameters when compared with the untreated control group.ConclusionsFindings from this study showed that MEJSL exhibited substantial anti-inflammatory actions in the in vitro and in vivo models. It also indicated that MEJSL anti-inflammatory mechanisms of action could be through interference with phase 2 inflammatory stressors, upregulation of cytoprotective genes, stabilization of inflammatory cell membranes and immunomodulatory activity.

Anti-inflammatory property and gas chromatography-mass spectrometry analysis of ethyl acetate fraction of Crateva adansonii DC leaves

Aims: This study was designed to determine the most active anti-inflammatory fraction of C. adansonii leaves and characterize the bioactive compounds. Methods: Dried and pulverized C. adansonii leaves (CAL) were extracted with 70% methanol, followed by successive solvent partitioning into hexane (CALH), ethyl acetate (CALE), butanol (CALB) and aqueous (CALA) leaf fractions. In vitro antioxidant assays carried out on the extract/fractions were DPPH, hydrogen peroxide, ferric reducing antioxidant power (FRAP), nitric oxide scavenging, total antioxidant capacity (TAC), total phenol (TP) and flavonoid (TF) assays. In vitro anti-inflammatory assays investigated were heat-induced bovine serum albumin (BSA) denaturation and human red blood cell (HRBC) membrane stabilization against hypotonicity- induced hemolysis. Formaldehyde-induced inflammation model in rats was carried out, followed by paw edema measurement as well as serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities assays. The ethyl acetate (CALE) fraction was subjected to GC-MS analytical method. Results: CAL extract and fractions substantially inhibited BSA denaturation and stabilized HRBC membrane against hypotonicity-induced hemolysis, however CALE exhibited the highest activity. CALE also suppressed formaldehyde-induced rat paw edema and significantly (p

Evaluation of anti-inflammatory and genotoxicity potentials of the fractions of &lt;i&gt;Archidium ohioense&lt;/i&gt; (Schimp. Ex Mull) extract

This study evaluated red blood cell (rbc) membrane stabilization and albumin denaturation, xanthine oxidase and lipoxygenase inhibitory activities of n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc), butanol (BuOH) and aqueous (Aq) fractions of Archidium ohioense methanolic extract using standard procedures. Diclofenac was used as a positive control for both rbc membrane stabilization and albumin denaturation studies while allopurinol and ascorbic acid respectively were used as positive control for xanthine oxidase and lipoxygenase inhibition measurements. Furthermore, genotoxic effect of the two most active fractions of the extract (EtOAc and DCM) on Allium cepa meristematic cells was investigated. Results showed that all the fractions inhibited heat-induced bovine serum albumin denaturation activity with the EtOAc and DCM fractions exhibiting better inhibitory activity than other fractions. Also, all the fractions except aqueous fraction compared favourably with the diclofenac (maximum stability of 86.94±0.00%) and protected stressed erythrocyte membrane at various concentrations tested. The maximum percentage erythrocyte stabilities were 97.39±0.00, 96.23±0.00, 91.85±0.00, 87.71±0.00 and 23.93±0.01% for A. ohioense EtOAc, DCM, n-Hex, BuOH and Aq fractions, respectively. It was observed that EtOAc fraction, DCM fraction and ascorbic acid inhibited lipoxygenase activity by 70.00±2.36, 75.00±1.67, and 62.50±8.84% respectively while 71.67±3.54, 50.00±0.00 and 77.50±1.76% of xanthine oxidase inhibition were elicited by EtOAc fraction, DCM fraction and allopurinol respectively. EtOAc and DCM fractions showed significant reduction in mitotic index and root lengths of Allium cepa at higher concentrations. Also, sticky chromosomes were observed for EtOAc (300 μg/ml) and DCM (300 -350 μg/ml) fractions. It was concluded that A. ohioense plant possesses antiinflammatory potential with the EtOAc and DCM fractions demonstrating very strong anti-inflammatory activity and compare favourably with standard reference drug (diclofenac). However, these fractions inhibited cell division of A. cepa cells at higher concentration. Keywords: Archidium ohioense , Lipoxygenase, Xanthine oxidase, Allium cepa , Genotoxicity, Mitotic index

Differentiation of glycated residue numbers on heat-induced structural changes of bovine serum albumin.

Glycation is an approach in dealing with heat-induced protein aggregation. The relationship between degree of glycation and heat-induced structural changes is still unclear. The present work investigates the effect of different numbers and sites of glycated residues on heat-induced structural changes of bovine serum albumin (BSA). Glycation of BSA was carried out with xylose (Xyl) and galactose (Gal) by Maillard reaction. Glycated residues in BSA were identified by liquid chromatography-tandem mass spectrometry, and heat-induced protein structural changes were characterized by fluorescence emission and synchronous fluorescence spectra, 8-anilino-1-naphthalenesulfonic acid fluorescence, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. The numbers of glycated residues were 2 and 13 when BSA was glycated by Gal (BSA - Gal) and Xyl (BSA - Xyl), respectively. There were shifts of maximum wavelengths and decreases in fluorescence intensities for both intrinsic and extrinsic fluorescences; shifts of FTIR amide I, III, and A bands; and decrease or increase of CD band intensities, α-helix and β-sheet percentages when BSA was heated. Glycation with Gal or Xyl restrained in similar degrees those changes, including fluorescence wavelengths, amide I band, CD band intensities, and α-helix and β-sheet percentages. Xyl glycated more residues than Gal, while their effects were similar in restraining heat-induced BSA structural changes. © 2017 Society of Chemical Industry.

Antioxidant and anti-inflammatory properties of selected polyherbal preparations: Oroki herbal, Swedish bitters and Yoyo bitters

Objective: Polyherbal preparations are extracts obtained from different medicinal plants. These herbal preparations are orally administered as therapy against numerous oxidative stress and inflammation mediated diseases. This study was designed to evaluate the antioxidant and anti-inflammatory properties of Oroki herbal, Swedish bitters and Yoyo bitters using in vitro models. Methods: Antioxidant studies were carried out using total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS), ferric reducing antioxidant power (FRAP) and 1’1’-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays while anti-inflammatory studies were performed using human red blood cell (HRBC) membrane stabilization and anti-denaturation of bovine serum albumin (BSA) assays. Results: Data showed that Oroki herbal had the highest TAC, inhibition of TBARS, FRAP absorbance range, DPPH scavenging activity while Swedish bitters had the highest stabilization of hypotonicity-induced hemolysis of HRBC and inhibition of heat-induced BSA denaturation when compared with other herbal preparations. Conclusion: Findings from this study indicated that Oroki herbal exhibited the highest antioxidant activity while Swedish bitters had the highest anti-inflammatory property in vitro. Furthermore, the medicinal properties of these polyherbal preparations could be attributed to their antioxidant and anti-inflammatory activities that could mitigate against oxidative stress and inflammation mediated diseases.

Influence of protein conformation on frictional properties of poly (vinyl alcohol) hydrogel for artificial cartilage

Poly (vinyl alcohol) (PVA) hydrogel is one of the anticipated materials for artificial cartilage. In our previous studies, wear of PVA hydrogel depended on content of proteins in lubricants. The secondary structures of bovine serum albumin (BSA) and human gamma globulin (HGG) were investigated in circular dichroism spectroscopy to clarify the influence of the proteins on frictional properties. BSA and HGG were mainly composed of the α-helix and the β-sheet, respectively. BSA containing the α-helix structure showed low friction compared to HGG composed of the β-sheet structure in mixed or boundary lubrication mode. The α-helix structure forms low shear layer because the α-helix structure is easily released from surfaces and low cohesive strength. HGG forms uniform adsorption layer, but showed higher friction than BSA in the rubbing with single protein. In the repeated rubbing with changing of lubricants from HGG to BSA, however, the final friction was reduced, because an optimum layered structure of proteins was formed. Hence, layered structure of proteins appears to play an important role to protect rubbing surfaces and to reduce friction. In heat treatment tests, heat-induced BSA showed very low friction because of reduction of the α-helix structure. Heat-induced HGG did not show large differences from native HGG, but could not bring low friction with heat-induced BSA. Thus it was shown that the protein conformation has effective influences on friction.

Fractal analysis of aggregates in heat-induced BSA gels

The fractal structure of the aggregates in heat-induced bovine serum albumin (BSA) gels was analyzed. Three kinds of BSA gels were prepared by heating BSA solutions: (i) pH 5.1 without CaCl 2; (ii) pH 7.0 with 30 mmol/dm 3 CaCl 2; and (iii) pH 7.0 with 5 mmol/dm 3 CaCl 2. First, from the concentration dependences of elasticity for the gels, using the theory of Shih et al. on colloidal gels, the fractal dimensions were evaluated. These BSA gels showed weak-link behavior according to the theory of Shih et al.: the limit of linearity γ 0 increased with increasing BSA concentration. Second, from the images of the aggregates in the gels obtained with confocal laser scanning microscopy, the fractal dimensions of heat-induced BSA gels were evaluated using the box counting method. The fractal dimensions D f for the three gels evaluated from rheological measurement were close to those evaluated from image analysis, the value of D f for the gels being ∼2.6–2.8.